Technical Field
The disclosure relates to the fixation of tissue samples for subsequent studies such as histopathology analysis.
Background Art
Biological tissue specimens are collected in many different circumstances and for a variety of scientific and medical purposes. One category of tissue specimens is collected for medical research and diagnostics of both humans and animals. An example subcategory is human tumor samples for oncology related studies.
Historically, tissue samples have been fixed and stained with a variety of chemicals to produce colorimetric indications of various features in the tissue. In medical diagnostics, this is generally referred to as histopathology. The basic process involves staining with dyes to produce contrasting colors for cellular elements. An example is Hematoxylin which is used to stain cell nuclei blue, plus eosin to stain cytoplasm and the extracellular matrix pink (called an H&E stain). Layered onto these background stains are histochemical reaction products e.g. to detect excess iron or copper and immunohistochemical staining procedures used to ascertain the presence of specific biomolecules e.g. proteins, specific nucleic acid structures, etc.
To the foregoing, medical science is adding a new dimension to histopathology to support what is broadly termed “personalized medicine” or “molecular medicine.” These broad terms encompass current histopathology techniques, in particular immunohistochemistry. However a key component of the new histopathology paradigm is the analysis of specific biomolecules in cells, specifically deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) and proteins and/or post-translationally modified proteins involved in cellular processes. DNA is most often associated with characteristic genetic changes in certain cancers and inherited genetic diseases. The presence and condition of RNA is a downstream indicator showing for example how active genes are in cells. Much of personalized medicine focuses on gene activity as reflected by RNA. The presence, absence, or quantitative amounts of RNA, and the relative amounts of specific RNAs to each other, all serve to form an “expression profile” or “gene expression signature.” The measurement of proteins involved in intra-cellular signaling, and specifically post-translational modifications to proteins, such as their phosphorylation status can provide critical information on the behavior of specific cell populations.
The use of gene expression profiling in personalized medicine is no longer a technology of the distant future. An example is the AGENDIA® MAMMAPRINT®, an RNA expression profile diagnostic approved by the FDA for determining the risk of long term breast cancer metastasis. See generally, van't Veer L J Dai H, van de Vijver M J, et al. (2002). “Gene expression profiling predicts clinical outcome of breast cancer”. Nature 415 (6871): 530-6. doi:10.1038/415530a; U.S. FDA 510(k) approval number K062694. As discussed for the AGENDIA® MAMMAPRINT® in its product literature, RNA integrity is critical for the effective use of expression profiling technology. The MAMMAPRINT® U.S. FDA label requires therefore a minimum level of RNA integrity.
Tissue specimens are archived for standard histopathology study using formalin-fixation with paraffin embedding (FFPE). FFPE damages RNA to a degree that archived tissue normally cannot be used for expression profile tests such as MAMMAPRINT® without great difficulty. There have however been some tests developed where the expression profile can be determined despite the poor quality of FFPE extracted RNA. See, e.g. Gray, Richard G., et al. “Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer.” Journal of Clinical Oncology 29.35 (2011): 4611-4619 (commercially sold as the Oncotype DX® colon cancer assay). These current FFPE extracted RNA assays rely on small RNA fragments, detected by hybridization or very short PCR amplicons, both of which limit the genes that can be evaluated. Even existing FFPE extracted RNA based tests could be improved by providing better RNA from FFPE tissue samples. Currently, RNA must be maintained by an alternative treatment of the tissue than FFPE such as the RNA extraction systems sold under the RNAlater® and RNAretain® trademarks and described in U.S. Pat. No. 6,204,375 and subsequent patents. These alternative RNA preservation techniques are not retroactively applicable and have not been successfully adopted for clinical applications.
A problem presented by the emergence of molecular diagnostics of RNA in tissue samples is the integration of such RNA testing with standard archival fixation of tissues samples. Any integrated change should not affect current histopathology procedures such as H&E staining.
Efforts to solve this integration problem have primarily focused of replacing FFPE with more RNA friendly fixation chemistries that are simultaneously compatible with current histopathology stains. One example is the HOPE tissue fixation technique. Jürgen Olert, et al., “HOPE-fixation: A novel fixing method and paraffin embedding technique for human soft tissues” (2001) Pathol Res Pract 197:823-826. Other RNA preserving tissue fixation chemistries have been offered commercially (e.g. the PAXgene® Tissue System).
HOPE tissue fixation and other techniques have not replaced FFPE in standard tissue sample preservation procedures, despite being available for over a decade. The problem to be solved then may be defined as providing a way to modify current FFPE procedures to enhance RNA stability with as little effect as possible on the FFPE process and the histopathology staining of the resulting fixed tissues. Ideally, the FFPE procedure modifications may be easily integrated into existing procedures and equipment and at a cost that does not de facto prohibit implementation.
RNA degradation in FFPE procedures has been studied thoroughly and described in the art recognized authoritative papers on the subject. Stephen M. Hewitt, Fraser A. Lewis, Yanxiang Cao, Richard C. Conrad, Maureen Cronin, Kathleen D. Danenberg, Thomas J. Goralski, John P. Langmore, Rajiv G. Raja, P. Mickey Williams, John F. Palma, and Janet A. Warrington, “Tissue Handling and Specimen Preparation in Surgical Pathology: Issues Concerning the Recovery of Nucleic Acids From Formalin-Fixed, Paraffin-Embedded Tissue”, Archives of Pathology & Laboratory Medicine 2008 132:12, 1929-1935; Joon-Yong Chung, Till Braunschweig, Reginald Williams, Natalie Guerrero, Karl M. Hoffmann, Mijung Kwon, Young K. Song, Steven K. Libutti, Stephen M. Hewitt, “Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue”, J Histochem Cytochem. 2008 November; 56(11): 1033-1042. doi: 10.1369/jhc.2008.951863.
While much is unknown regarding the factors at play in RNA degradation, the RNA degradation profile during FFPE is generally known as summarized in FIG. 1. As can be seen, once the tissue specimen is fixed and embedded, the RNA degrades only very slowly over time. The bulk of RNA degradation occurs during FFPE processing and fixation. The mechanisms of RNA degradation pre- and post-processing (i.e. post paraffin embedding) are distinct. For example, the chemical modifications of RNA by Formalin during fixation are known to involve RNA fragmentation and chemical crosslinking to proteins and other biomolecules in the tissues.
FFPE processes are diverse in various aspects. The foregoing papers have surveyed and identified some optimal FFPE conditions and best practices for RNA stability such as the use of phosphate buffered Formalin over other buffer systems. However, these optimized practices alone will often not be sufficient to produce fixed tissues that are viable RNA sources for gene expression profile studies. The following disclosure therefore provides a solution to the foregoing problem(s) of poor RNA stability during FFPE procedures.